Bottom Line:
Equally important, we show Mtb induces the early expression of miR-142-3p and partially down-regulates N-Wasp protein levels in both the murine J774A.1 cell line and primary human Mφs.As proof of principle, the partial siRNA-mediated knock down of N-Wasp resulted in a decrease of Mtb intake by human Mφs, reflected in lower levels of colony-forming units (CFU) counts over time.We therefore propose the modulation of miRNAs as a novel strategy in mycobacterial infection to control factors involved in actin filament assembly and other early events of phagolysosome biogenesis.

ABSTRACTMycobacterium tuberculosis (Mtb) is a successful intracellular pathogen that thrives in macrophages (Mφs). There is a need to better understand how Mtb alters cellular processes like phagolysosome biogenesis, a classical determinant of its pathogenesis. A central feature of this bacteria's strategy is the manipulation of Mφ actin. Here, we examined the role of microRNAs (miRNAs) as a potential mechanism in the regulation of actin-mediated events leading to phagocytosis in the context of mycobacteria infection. Given that non-virulent Mycobacterium smegmatis also controls actin filament assembly to prolong its intracellular survival inside host cells, we performed a global transcriptomic analysis to assess the modulation of miRNAs upon M. smegmatis infection of the murine Mφ cell line, J774A.1. This approach identified miR-142-3p as a key candidate to be involved in the regulation of actin dynamics required in phagocytosis. We unequivocally demonstrate that miR-142-3p targets N-Wasp, an actin-binding protein required during microbial challenge. A gain-of-function approach for miR-142-3p revealed a down-regulation of N-Wasp expression accompanied by a decrease of mycobacteria intake, while a loss-of-function approach yielded the reciprocal increase of the phagocytosis process. Equally important, we show Mtb induces the early expression of miR-142-3p and partially down-regulates N-Wasp protein levels in both the murine J774A.1 cell line and primary human Mφs. As proof of principle, the partial siRNA-mediated knock down of N-Wasp resulted in a decrease of Mtb intake by human Mφs, reflected in lower levels of colony-forming units (CFU) counts over time. We therefore propose the modulation of miRNAs as a novel strategy in mycobacterial infection to control factors involved in actin filament assembly and other early events of phagolysosome biogenesis.

Mentions:
The reduction of N-Wasp expression by the treatment mimicking miR-142-3p of Mφs infected with either mycobacterial strain, prompted us to investigate for a possible role for this miRNA in controlling the early stages of phagocytosis. To assess this, we again employed the use of either “mimics” or “inhibitors” of miR-142-3p activity. Our hypothesis predicted a decrease in bacterial intake with the use of the mimics and an increase in the presence of inhibitors. As depicted in Figure 3A, confocal analysis confirmed that Mφs treated with the mimics resulted in a reduced amount of intracellular M. smegmatis at 4 hpi when compared to the negative control (scrambled miRNA). By contrast, Mφs treated with the inhibitors led to a dramatic increase in the amount of intracellular M. smegmatis. This effect was accompanied by distinct morphological changes in terms of cell size and large numbers of phagocytic cups, as compared to Mφs treated with the mimics (Figure 3B). The quantification analysis of the confocal analysis is illustrated in Figure 3C. Furthermore, the bacterial intake under these conditions was also measured at 1 or 4 hpi by CFU assays; this alternative quantitative method revealed similar effects to those obtained by confocal microscopy (Figure 3D). In the case for Mtb, Mφs treated with the miR-142-3p mimics resulted in a significant reduced amount of intracellular Mtb when compared to the miRNA control at 4 hpi, thus confirming our previous results with M. smegmatis challenge (Figure 4A). However, we were surprised that treatment of Mφs with the inhibitors did not lead to a significant increased level of intracellular Mtb, as it was the case for M. smegmatis (Figure 4B). The quantification analysis of the confocal analysis is provided in Figure 4C.

Mentions:
The reduction of N-Wasp expression by the treatment mimicking miR-142-3p of Mφs infected with either mycobacterial strain, prompted us to investigate for a possible role for this miRNA in controlling the early stages of phagocytosis. To assess this, we again employed the use of either “mimics” or “inhibitors” of miR-142-3p activity. Our hypothesis predicted a decrease in bacterial intake with the use of the mimics and an increase in the presence of inhibitors. As depicted in Figure 3A, confocal analysis confirmed that Mφs treated with the mimics resulted in a reduced amount of intracellular M. smegmatis at 4 hpi when compared to the negative control (scrambled miRNA). By contrast, Mφs treated with the inhibitors led to a dramatic increase in the amount of intracellular M. smegmatis. This effect was accompanied by distinct morphological changes in terms of cell size and large numbers of phagocytic cups, as compared to Mφs treated with the mimics (Figure 3B). The quantification analysis of the confocal analysis is illustrated in Figure 3C. Furthermore, the bacterial intake under these conditions was also measured at 1 or 4 hpi by CFU assays; this alternative quantitative method revealed similar effects to those obtained by confocal microscopy (Figure 3D). In the case for Mtb, Mφs treated with the miR-142-3p mimics resulted in a significant reduced amount of intracellular Mtb when compared to the miRNA control at 4 hpi, thus confirming our previous results with M. smegmatis challenge (Figure 4A). However, we were surprised that treatment of Mφs with the inhibitors did not lead to a significant increased level of intracellular Mtb, as it was the case for M. smegmatis (Figure 4B). The quantification analysis of the confocal analysis is provided in Figure 4C.

Bottom Line:
Equally important, we show Mtb induces the early expression of miR-142-3p and partially down-regulates N-Wasp protein levels in both the murine J774A.1 cell line and primary human Mφs.As proof of principle, the partial siRNA-mediated knock down of N-Wasp resulted in a decrease of Mtb intake by human Mφs, reflected in lower levels of colony-forming units (CFU) counts over time.We therefore propose the modulation of miRNAs as a novel strategy in mycobacterial infection to control factors involved in actin filament assembly and other early events of phagolysosome biogenesis.

ABSTRACTMycobacterium tuberculosis (Mtb) is a successful intracellular pathogen that thrives in macrophages (Mφs). There is a need to better understand how Mtb alters cellular processes like phagolysosome biogenesis, a classical determinant of its pathogenesis. A central feature of this bacteria's strategy is the manipulation of Mφ actin. Here, we examined the role of microRNAs (miRNAs) as a potential mechanism in the regulation of actin-mediated events leading to phagocytosis in the context of mycobacteria infection. Given that non-virulent Mycobacterium smegmatis also controls actin filament assembly to prolong its intracellular survival inside host cells, we performed a global transcriptomic analysis to assess the modulation of miRNAs upon M. smegmatis infection of the murine Mφ cell line, J774A.1. This approach identified miR-142-3p as a key candidate to be involved in the regulation of actin dynamics required in phagocytosis. We unequivocally demonstrate that miR-142-3p targets N-Wasp, an actin-binding protein required during microbial challenge. A gain-of-function approach for miR-142-3p revealed a down-regulation of N-Wasp expression accompanied by a decrease of mycobacteria intake, while a loss-of-function approach yielded the reciprocal increase of the phagocytosis process. Equally important, we show Mtb induces the early expression of miR-142-3p and partially down-regulates N-Wasp protein levels in both the murine J774A.1 cell line and primary human Mφs. As proof of principle, the partial siRNA-mediated knock down of N-Wasp resulted in a decrease of Mtb intake by human Mφs, reflected in lower levels of colony-forming units (CFU) counts over time. We therefore propose the modulation of miRNAs as a novel strategy in mycobacterial infection to control factors involved in actin filament assembly and other early events of phagolysosome biogenesis.